NI = the

number of intercepts that cross basal membrane,

number of intercepts that cross basal membrane, which is proportional to the perimeter of the airway; L = number of points hitting the airway lumen, which is proportional to the intraluminal area. BI was quantified in five non-cartilaginous airways per animal at 400× magnification ( Sakae et al., 1994). Airway smooth muscle area, airway epithelium thickness and edema were defined as the learn more number of point hitting, respectively, in smooth muscle and epithelial cells and peribronchial edema. This value was divided by the number of intercepts that cross the basal membrane, which is proportional to the perimeter of the airway (Sakae et al., 1994 and Vieira et al., 2007). Measurements were performed in five airways per animal at 1000× magnification. After blood collection from the cava vein, the samples were immediately centrifuged for 15 min (5 °C; 1000 rpm). Serum samples were stored at −70 °C until the Selleck Everolimus assay was performed. A PCA reaction was used to detect and estimate the levels of anaphylactic IgE and IgG1 OVA-specific antibodies as previously described (Ovary, 1964 and Mota and Perini, 1970). Briefly, the back of a naïve guinea

pig was shaved, and 0.1 ml of different serum dilutions was injected intradermally. Thirty naïve guinea pigs were used to evaluate the PCA, and the serum from each animal was included in the study (n = 30). After a long latent period of 48 h for IgE or a short period of 24 h for IgG1, the animals were challenged intravenously (i.v.) with 1 ml of a 0.5% solution of Evans blue in saline (0.9% NaCl) containing 1 mg of antigen (ovalbumin). The animals were

euthanized 30 min after injection of the antigen, and the diameters of the blue spots on the inner surface of the flayed skin were measured. To detect the IgG1-type antibody, the serum was heated for 3 h at 56 °C to inactivate IgE activity; the heated serum was injected for PCA after a short latency period. The PCA titers were defined as the highest dilutions that gave an intradermal allergic about reaction larger than 5 mm in diameter in triplicate tests ( Ovary, 1964 and Mota and Perini, 1970). One-way analysis of variance (ANOVA) followed by a Student–Newman–Keuls post hoc test (parametric data) or ANOVA on ranks followed by Dunn’s post hoc test (non-parametric data) were used to compare the different parameters between groups. The values were expressed as the mean ± SD for parametric data and as the median (variance) for non-parametric data. The level of significance was set at p